ALBERT

Description: Abstract 1743 Poster Board I-769 Background Molecular targeting drugs, all-trans retinoic acid (ATRA)and arsenic trioxide (ATO), have major advances in the treatment of acute promyelocytic leukemia (APL). However, resistance to these drugs has been also observed in clinical practice. ATRA acts as a ligand for retinoic acid receptor alpha (RAR) and restores the aberrant transcription repression by PML-RARA fusion protein in APL cells. Previous reports demonstrate that amino-acids substitution, resulting from genetic mutations, in ligand binding domain (LBD) of RARA region of PML-RARA were closely related to drug resistance to ATRA therapy. In contrast, for ATO therapy, the molecular mechanisms of the effectiveness and also the resistance are still unclear. Here we identified a PML-RARA that holds double genetic missense mutations in RARA and PML regions, respectively, from an APL patient, who showed clinically resistance to ATRA and ATO therapy. These mutations were observed as his disease progression, and we are interested in the relationship between these mutations with drug resistance to ATRA and/or ATO. Aims Analyses of the molecular and clinical significance of the double missense mutations of PML-RARA for disease progression and resistance to ATRA and ATO therapy. Results Eight APL patients were treated with ATO in Nagoya University Hospital, Japan, during ∼5 years from Apr. 1, 2000 to Dec. 31, 2004. One out of 8 patients showed clinically ATO resistance. The patient showing ATO resistance firstly diagnosed as APL (M3 variant) from cytogenetic and chromosomal analyses, and complete remission was obtained after combination chemotherapy with ATRA. Molecular CR was confirmed by RT-PCR analysis, but after 3 month from the induction therapy, ATRA-resistant relapse was observed. After treatment with ATO therapy, response was observed, but the effectiveness was gradually decreased, resulting finally into the resistance. The patient died of disease progression. During his 7 years clinical course, leukemia cells were harvested repeatedly from his bone marrow and peripheral blood. RT-PCR using the total RNA from his tumor cells followed by DNA sequencing was performed, with the result of PML-RARA fusion gene with the bcr3 breakpoint in the intron 3 of PML. When using the tumor cells that were harvested at his terminal stage, a missense point mutation in the LBD of the RARA region of PML-RARA was confirmed. Furthermore, missense point mutation in the PML-B2 domain was also confirmed in the same cDNA clones. Interestingly, these mutations were not observed in the leukemia cells obtained at the onset. These mutations were analyzed in each sample that was obtained as his disease progressed, and some correlation between disease progression and/or the drug resistance and the timing of appearance of these two mutations were suggested. These mutated fusion transcripts were cloned into expression vectors, and we are now analyzing the function relating to the drug resistance and disease progression. Conclusions Double genetic missense mutations in the RARA-LBD and PML-B2 of PML-RARA were confirmed in ATRA and ATO resistant patient. These genetic mutations were confirmed in the leukemia cells during his disease progression, and the relationship between those mutations and drug resistances were suggested from the clinical features. Mutations in the PML-B2 domain has not been reported previously, thus, it may be important to show whether this type of mutations are related to the drug resistance, especially to ATO therapy. Disclosures Kiyoi: Novartis Pharma Co. Ltd.: Research Funding; Kyowa Hakko Kirin Co. Ltd.: Consultancy. Naoe:Kyowa Hakko Kirin Co., Ltd. : Research Funding; Chugai Pharmaceutical Co.,Ltd.: Research Funding; Wyeth K.K.: Research Funding.

Description: Arsenic trioxide (As2O3) is a highly effective treatment for patients with refractory/relapsed acute promyelocytic leukemia (APL), but resistance to As2O3 has recently been seen. In the present study, we report the findings that 2 of 15 patients with refractory/relapsed APL treated with As2O3 were clinically As2O3 resistant. Leukemia cells from these 2 patients harbored missense mutations in promyelocytic leukemia gene–retinoic acid receptor-α gene (PML-RARA) transcripts, resulting in amino acid substitutions of A216V and L218P in the PML B2 domain. When wild-type or mutated PML-RARA (PR-WT and PR-B/L-mut, respectively) were overexpressed in HeLa cells, immunoblotting showed SUMOylated and/or oligomerized protein bands in PR-WT but not in PR-B/L-mut after As2O3 treatment. Protein-localization analysis indicated that PR-WT in the soluble fraction was transferred to the insoluble fraction after treatment with As2O3, but PR-B/L-mut was stably detected in fractions both with and without As2O3. Immunofluorescent microscopy analysis showed PR-WT localization as a microgranular pattern in the cytoplasm without As2O3 and as a macrogranular pattern with As2O3. PR-B/L-mut was diffusely observed in the cytoplasm with and without As2O3. Nearly identical localization patterns were observed in patients' primary cells. Therefore, B2 domain mutations may play an important role in aberrant molecular responses to As2O3 and may be critical for As2O3 resistance in APL.

Description: PML-RARα is a chimeric transcription factor deeply associated with acute promyelocytic leukemia (APL). PML-RARα plays an important role in the aberrant transcription repression on the target genes of wild type retinoic acid receptors (RARα). Pharmacological concentration of all-trans retinoic acid (ATRA) induces transcription de-repression on several target genes, and results in terminal differentiation of APL cells. However, the detailed mechanisms of transcription repression by PML-RARα in vivo are still unclear. Here we demonstrated that histone deacetylase 3 (HDAC3), one component of the N-CoR (nuclear receptor co-repressor)-TBL1/R1 (transducin beta-like protein 1/relating protein) transcription repressor protein complex, is a key regulator of the transcription repression by PML-RARα in vivo. Using immunoprecipitation (IP) assay, we first demonstrated that PML-RARα physically interacted with N-CoR/HDAC3 in vivo in the absence of ligand. The interaction was dissociated by adding ATRA in the dose dependent manner. Next we showed, using chromatin immunoprecipitation (ChIP) assay, that N-CoR/HDAC3 co-repressor complex was recruited to the endogenous target gene promoters (RARβ and CYP26) through PML-RARα. The neighboring histone H4 was de-acetylated and the gene expression was significantly repressed. When HDAC3 protein is knocked down by RNA interference in PML-RARα-presenting cells, the endogenous target gene expression was significantly activated. Almost the same results were also obtained when performing the luciferase reporter assay using RARβ and CYP26 promoter reporter vectors. Previously, we have shown that N-CoR-TBLR1 is recruited to the target gene promoter through PML-RARα in the absence of ligand, resulting in the transcription repression. Consistent with these data, it is strongly suggested that N-CoR/HDAC3/TBLR1 co-repressor complex is closely related to the aberrant transcription regulation by PML-RARα in APL cells. Furthermore, we also confirmed that PLZF-RARα, which is expressed in ATRA resistant APL cells, interacted with N-CoR/HDAC3/TBLR1 in ligand independent manner. These insights provide not only the basic mechanism of transcription repression by leukemia-related chimeric transcription factors, but also the new molecular targets for the transcription therapy for leukemia.